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At least 415 records · Page 23Linked to original sources
PubMed2007

Evaluation of viral removal by nanofiltration using real-time quantitative polymerase chain reaction.

Nanofiltration is commonly introduced into purification processes of biologics produced in mammalian cells to serve as a designated step for removal of potential exogenous viral contaminants and endogenous retrovirus-like particles. The LRV (log reduction value) achieved by nanofiltration is often determined by cell-based infectivity assay, which is time-consuming and labour-intensive. We have explored the possibility of employing QPCR (quantitative PCR) to evaluate LRV achieved by nanofiltration in scaled-down studies using two model viruses, namely xenotropic murine leukemia virus and murine minute virus. We report here the successful development of a QPCR-based method suitable for quantification of virus removal by nanofiltration. The method includes a nuclease treatment step to remove free viral nucleic acids, while viral genome associated with intact virus particles is shielded from the nuclease. In addition, HIV Armored RNA was included as an internal control to ensure the accuracy and reliability of the method. The QPCRbased method described here provides several advantages such as better sensitivity, faster turnaround time, reduced cost and higher throughput over the traditional cell-based infectivity assays.

Leukemia Virus, Murine↗
PubMed2003

Different frequencies of Streptococcus anginosus infection in oral cancer and esophageal cancer.

Multiple cancers frequently occur in the upper aerodigestive tract. The high incidence rate of multiple carcinomas in this region is often explained in terms of involvement of the same underlying risk factors. It has been reported that the oral bacterium Streptococcus anginosus (S. anginosus) is associated with esophageal, gastric, and pharyngeal cancer tissues. In this study, a highly specific quantification method for S. anginosus DNA using real-time PCR was established. We employed this assay to determine whether S. anginosus is also associated with oral cancer tissues. This precise quantification method revealed different degrees of infection with S. anginosus in esophageal cancer and oral cancer. We assayed 10 ng of genomic DNA from cancer tissues, and found that eight of 18 samples (44%) from the esophagus contained a detectable level (>10 fg) of S. anginosus DNA, whereas this was the case for only five of 38 samples (13%) of oral cancer. The quantity of S. anginosus DNA in the esophageal cancer tissues was significantly higher than in oral cancer. The maximum amount of S. anginosus DNA was approximately ten times higher in esophageal than in oral cancer tissues. In addition, none of the five different oral cancer sites (floor of the mouth, mandibular gingival, maxillary gingival, buccal mucosal, and tongue) showed significant signs of S. anginosus infection. On the other hand, most non-cancerous tissues of the esophagus and tongue showed an undetectable level of S. anginosus. These results suggest that S. anginosus is associated with esophageal cancer, but is not closely related with oral cancer.

Base Sequence↗
PubMed2006

Real-time PCR detection and quantification of vector trichodorid nematodes and Tobacco rattle virus.

This report describes a novel diagnostic method for virus-vector trichodorid nematodes and associated Tobacco rattle virus (TRV) based on a real-time fluorogenic 5' nuclease PCR assay (TaqMan). Two independent primer/probe sets were designed targeting the 18S gene of the ribosomal cistron for the trichodorid species, Paratrichodorus pachydermus and Trichodorus similis. Assays using purified plasmid DNA containing clones of the 18S region and genomic DNA extracted from individuals from both nematode species displayed high specificity as no cros s-reaction was observed between the species or with two non-target trichodorid species Paratrichodorus anemones and Trichodorus primitivus. Relative quantification of target DNA present in unknown samples was performed by comparison of the fluorescence signals of the samples to those obtained from plasmid standard dilutions. Three primer/probe sets were also used to target TRV; one set for RNA1 and the two other sets for RNA2 of specific isolates (TRV-PpK20 and TRV-TpO1). Detection of both trichodorid species and TRV RNA1 and RNA2 from a single sample was achieved and field samples were used to demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular information in relation to risk assessment in the field.

Analysis of Variance↗
PubMed1987

A search for DNA alterations in the aging mammalian genome: an experimental strategy.

In order to experimentally test the hypothesis that at the basal level senescence is caused by instabilities in DNA, techniques are required that allow the sensitive detection, quantification and characterization of DNA damage and DNA sequence changes in various organs and tissues of naturally aging mammals. In this article a strategy is presented that should allow the analysis of DNA damage metabolism in aging mammals from the original changes in the chemical structure of DNA (DNA damages) via their processing (DNA repair) to the molecular endpoints in terms of gene mutations and DNA rearrangements. With respect to the detection of DNA damage, a short overview is provided of recently emerged biochemical and immunochemical methods that can be applied immediately to study the spectrum of DNA damages in various organs and tissues of aging animals or humans. By contrast, before one is able to study low frequency changes in DNA sequence organization in somatic tissues, formidable problems have yet to be overcome. This is mainly due to the fact that the scale of highly advanced recombinant DNA techniques recently developed is almost totally devoted to the analysis of heritable genetic factors. In order to be able to study low frequency changes in DNA sequence organization in somatic tissues, we have initiated experimental approaches based on recently emerged shuttle vector technology in combination with transgenic mice, and on new electrophoretic separation principles. These approaches and their potentialities for testing somatic mutation theories of aging are discussed.

Aging↗
PubMed2002

Assigning probes into a small number of pools separable by electrophoresis.

MOTIVATION: Measuring transcriptional expression levels (transcriptional profiling) has become one of the most important methods in functional genomics. Still, new measuring methods are needed to obtain more reliable, quantitative data about transcription on a genomic scale. In this paper we concentrate on certain computational optimization problems arising in the design of one such novel method. From a computational point of view the key feature of the new method is that the hybridized probes are distinguished from each other based on their different size. Therefore the probes have to be assigned into pools such that the probes in the same pool have unique sizes different enough from each other. Identification of expressed RNA is given by probe pool and probe size while quantification is given by the label of the probe, e.g. fluorescence intensity. RESULTS: We show how to computationally find the probes and assign them into pools for a whole genome such that (i) each gene has a specific probe suitable for amplification and hybridization, and (ii) the expression level measurement can be done in a minimal number of pools separable by electrophoresis in order to minimize the total experiment cost of the measurement. Our main result is a polynomial-time approximation algorithm for assigning the probes into pools. We demonstrate the feasibility of the procedure by selecting probes for the yeast genome and assigning them into less than 100 pools. The probe sequences and their assignment into pools are available for academic research on request from the authors.

Algorithms↗
PubMed2026

Dogme: a nextflow pipeline for reprocessing nanopore RNA and DNA modifications.

MOTIVATION: Oxford Nanopore (ONT) sequencing allows for the direct detection of RNA and DNA modifications from unamplified nucleic acids, which is a significant advantage over other platforms. However, the rapid updates to ONT basecalling models and the evolving landscape of computational tools for modification detection bring about challenges for reproducible and standardized analyses. To address these challenges, we developed Dogme to automate basecalling, alignment, modification detection, and transcript quantification. Dogme automates the reprocessing of ONT POD5 files by integrating basecalling using Dorado, read mapping using minimap2 and subsequent analysis steps such as running modkit. The pipeline supports three major types of sequencing data-direct RNA (dRNA), complementary DNA (cDNA), and genomic DNA (gDNA). Dogme facilitates detection of diverse RNA modifications supported by Dorado such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), inosine, pseudouridine, 2'-O-methylation (Nm) and DNA methylation, while concurrently quantifying full-length transcript isoforms LR-Kallisto for transcript quantification for dRNA and cDNA. RESULTS: We applied Dogme to three separate mouse C2C12 myoblast replicates using direct RNA sequencing on MinION flow cells. We detected 96 603 m6A, 43 476 m5C, 8829 inosine, 10 055 pseudouridine, and 30 320 Nm sites in three biological replicates. The pipeline produced reproducible modification profiles and transcript expression levels across replicates, demonstrating its utility for integrative long-read transcriptomic and epigenomic analyses. AVAILABILITY AND IMPLEMENTATION: Dogme is implemented in Nextflow and is freely available under the MIT license at https://github.com/mortazavilab/dogme, with documentation provided for installation and usage.

RNA↗
PubMed2006

Identification and functional analysis of a human homologue of the monkey replication origin ors8.

We previously isolated from African green monkey (CV-1) cells a replication origin, ors8, that is active at the onset of S-phase. Here, its homologous sequence (hors8, accession number: DQ230978) was amplified from human cells, using the monkey-ors8-specific primers. Sequence alignment between the monkey and the human fragment revealed a 92% identity. Nascent DNA abundance analysis, involving quantification by real-time PCR, indicated that hors8 is an active replication origin, as the abundance of nascent DNA from a genomic region containing it was 97-fold higher relative to a non-origin region in the same locus. Furthermore, the data showed that the hors8 fragment is capable of supporting the episomal replication of its plasmid, when cloned into pBlueScript (pBS), as assayed by the DpnI resistance assay after transfection of HeLa cells. A quantitative chromatin immunoprecipitation (ChIP) assay, using antibodies against Ku, Orc2, and Cdc6, showed that these DNA replication initiator proteins were associated in vivo with the human ors8 (hors8). Finally, nascent DNA abundance experiments from human cells synchronized at different phases of the cell cycle revealed that hors8 is a late-firing origin of DNA replication, having the highest activity 8 h after release from late G(1).

Animals↗
PubMed2006

Role of endogenous retroviruses in autoimmune diseases.

Molecular epidemiologic proof that HERVs and other retroelements are involved in autoimmunity or other disorders is complicated by their large numbers in the human genome. As discussed, most HERVs are no longer functional or active because of the accumulation of mutations, frameshifts, and deletions. Detection or quantification of HERV transcripts that may be pathologically involved in a particular autoimmune disease thus is often compromised by the presence in great excess of related, but nonfunctional, RNA. This phenomenon should not deter active work in the field, although it will require development of improved methods to discriminate accurately between closely related RNA transcripts. Development of improved immunologic methods to precisely identify epitopes on autoantigens or rare self-reactive T-cell clones may further implicate HERVs and the other repetitive elements in regulation of the immune system in health and disease.

Animals↗
PubMed1994

Fine mapping of a replication origin of human DNA.

A highly sensitive procedure was developed for the identification of the origin of bidirectional DNA synthesis in single-copy replicons of mammalian cells. The method, which does not require cell synchronization or permeabilization, entails the absolute quantification, by a competitive PCR procedure in newly synthesized DNA samples, of the abundance of neighboring DNA fragments distributed along a given genomic region. This procedure was utilized for mapping the start site of DNA replication in a 13.7-kb region of human chromosome 19 coding for lamin B2, which is replicated immediately after the onset of S phase in HL-60 cells. Within this region, DNA replication initiates in a 474-bp area corresponding to the 3' noncoding end of the lamin B2 gene and the nontranscribed spacer between this gene and the 5' end of another highly transcribed one. This localization was obtained both in aphidicolin-synchronized and in exponentially growing HL-60 cells.

Animals↗
PubMed2026

Multi-season analysis reveals hundreds of drought-responsive genes in sorghum.

Persistent drought affects global crop production and is becoming more severe in many parts of the world in recent decades. Deciphering how plants respond to drought will facilitate the development of flexible mitigation strategies. Sorghum bicolor L. Moench (sorghum), a major cereal crop and an emerging bioenergy crop, exhibits remarkable resilience to drought. To better understand the molecular traits that underlie sorghum's remarkable drought tolerance, we undertook a large-scale sorghum gene expression profiling effort, totaling nearly 1500 transcriptome profiles, across a 3-year field study with replicated plots in California's Central Valley. This study included time-resolved gene expression data from roots and leaves of two sorghum genotypes, BTx642 and RTx430, with different pre-flowering and post-flowering drought-tolerance adaptations under control and drought conditions. Quantification of genotype-specific drought tolerance effects was enabled by de novo sequencing, assembly, and annotation of both BTx642 and RTx430 genomes. These reference-quality genomes were used to construct a pangene set for characterizing conserved and genotype-specific expression. By integrating time-resolved transcriptomic responses to drought in the field across three consecutive years, we identified a set of 726 drought-responsive genes that responded similarly in all 3 years of our field study. Functional enrichment analysis identified abiotic stress, secondary cell wall-related processes and metabolism as particularly affected under both types of drought stress. We also found that some glyoxylate cycle pathway genes, including malate synthase and isocitrate lyase, are differentially regulated particularly during post-flowering drought stress, implicating this pathway as potentially important for drought responsiveness. This expansive dataset represents a unique resource for sorghum and drought research communities and provides a methodological framework for the integration of multi-faceted time-resolved transcriptomic datasets.

Sorghum↗
PubMed1997

Use of manganese in RT-PCR eliminates PCR artifacts resulting from DNase I digestion.

The precise quantification of rare mRNA copies from intronless genes by reverse transcription polymerase chain reaction (RT-PCR) requires the complete removal of genomic DNA because discrimination of cDNA and DNA amplification products by differing sizes of PCR products is not possible. Elimination of DNA is achieved by treating the RNA sample with RNase-free DNase I before RT-PCR. The lack of a PCR product from DNase-treated RNA samples before RT is usually accepted as a proof of efficient DNA destruction. However, this may vary depending on the metal cofactor used in the DNase I cleavage. Treating DNA-contaminated RNA samples with DNase I and magnesium as a cofactor creates a negative PCR control after digestion without further RT. Paradoxically, after additional RT-PCR, the original intron-containing DNA fragment size may be produced again. In the presence of manganese as cofactor, RT-created DNA fragments do not appear. This is because in the presence of manganese, DNase I cleaves both DNA strands at approximately the same site, yielding DNA fragments that are blunt-ended or that have protruding termini of only one or two nucleotides in length. However, overlapping fragments with the potential to recombine are created by DNase digestion with magnesium as cofactor. Because one cannot differentiate between a PCR signal produced by RNA and one produced by recombined DNA after DNase I digestion and RT, all such DNase I assays should be performed with manganese instead of magnesium.

Artifacts↗
PubMed2003

The use of real-time reverse transcriptase PCR for the quantification of cytokine gene expression.

Real-time reverse transcriptase polymerase chain reaction (RT-PCR) is becoming a widely used method to quantify cytokines from cells, tissues, or tissue biopsies. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in a single step. Using TaqMan chemistry (Applied Biosystems, Foster City, CA) and the ABI Prism 7700 Sequence Detection System (Applied Biosystems), we validated a large panel of murine and human cytokines, as we as other factors playing a role in the immune system, such a chemokines and apoptotic markers. Although the method allows fast, sensitive, and accurate quantification, different control assays are necessary for the method to be reliable. By construction of complementary DNA (cDNA) plasmid clones, standard curves are generated that allow direct quantification of every unknown sample. Furthermore, the choice of a reliable housekeeping gene is very important. Finally, co-amplification of contaminating genomic DNA is avoided by designing sets of primers located in different exons or or intron-exon junctions. In conclusion, the real-time RT-PCF technique is very accurate and sensitive, allows high through put, and can be performed on very small samples. The development of real-time RT-PCR has resulted in an exponential increase in its use over the last couple of years, and the method has undoubtedly become the standard for quantifying cytokine patterns, clarifying many functional properties of immune cells and their associated diseases.

Animals↗
PubMed1990

Structure of chicken aldolase C mRNA and its expression in the liver and brain during development.

Chicken aldolase B (liver-type) and C (brain-type) cDNAs were isolated and their nucleotide sequences determined. Southern blot analysis of chicken genomic DNA using the fragments of aldolase C cDNA suggested that the aldolase C gene is a single-copy gene. The quantification by Northern blot analysis of aldolase B and C mRNAs in the chicken liver and brain during development showed that in the liver, the B gene was progressively activated, while C gene expression was extinguished reciprocally. In the brain, the B gene was silent throughout the development, while the C gene was activated progressively, reaching a product level of more than 20-fold that of the 7-day-old embryo.

Amino Acid Sequence↗
PubMed2003

A specific cis-hairpin ribozyme facilitates infection of a TMV-based DNA vector in tobacco protoplasts.

The effect of a specific cis-hairpin ribozyme on TMV-based vectors in the infection of tobacco protoplasts was studied. Vectors contained full-length TMV genome cDNA linked to a T7 promoter or a CaMV 35S promoter at the 5'-end and an NOS gene polyadenylation signal at the 3'-end. The coat protein (CP) gene was replaced with the green fluorescent protein (GFPuv) gene allowing quantification of protoplast infection. In plasmids pTMVGFPRIB (T7-driven) and pSTMVGFPRIB (CaMV 35S-driven), the cDNA fragment of the cis-hairpin ribozyme (designed to specifically cleave the transcripts immediately downstream of the 3'-terminus of TMV RNA) was inserted between the 3'-terminus of TMV genome and NOS sequence. The in vitro transcript TMVGFPRIB was three- to fivefold more infectious than the control TMVGFPNOS. Northern blot analysis indicated that the 3'-terminal non-viral sequence had been cleaved from the in vitro transcripts by the cis-hairpin ribozyme soon after in vitro transcription. pSTMVGFPRIB and pSTMVGFPNOS plasmid DNAs were, as expected, less infectious than their in vitro transcript counterparts. However, pSTMVGFPRIB was somewhat more infectious than pSTMVGFPNOS. Northern blot analysis indicated that pSTMVGFPRIB synthesized more genomic and sub-genomic RNAs in the protoplasts. The significant increase in infectivity and viral RNA synthesis is due to the specific activity of the cis-hairpin ribozyme in vivo. Therefore, the cis-hairpin ribozyme described here may improve TMV-based vectors in the expression of foreign protein in plants.

Base Sequence↗
PubMed2002

Development and evaluation of an internally controlled semiautomated PCR assay for quantification of cell-free cytomegalovirus.

Quantification of circulating human cytomegalovirus (HCMV) is useful in clinical contexts such as virological surveillance of bone marrow transplant recipients and monitoring of antiviral therapy. This report describes an internally controlled, quantitative, semiautomated, HCMV genome assay that was developed primarily to measure HCMV DNA in the plasma of severely leucopaenic patients. It exhibits greater sensitivity, wider dynamic range and higher sample throughput than a number of previously described commercial and "in-house" assays. Viral DNA extraction from EDTA plasma samples was automated using a BioRobot 9604 (Qiagen). HCMV strain AD169 was used to prepare a calibration curve and murine cytomegalovirus (MCMV) strain Smith was added as internal control to all calibration standards and test samples. Amplification was performed using a set of primers based on the HCMV UL50 region, capable of amplifying both human and murine CMV. The yield of biotinylated polymerase chain reaction (PCR) products was estimated using HCMV-specific and MCMV-specific enzyme-labelled probes and automated chemiluminescence detection. Log-transformed HCMV-to-MCMV signal ratios were calculated and used for quantification of test samples against simultaneously extracted MCMV-spiked calibration standards. Evaluation of the assay sensitivity by Probit analysis demonstrated a 95% probability of detection at 100 HCMV genomes per ml of plasma; the dynamic range was shown to be > or = 4 log(10). A total of 315 samples from 61 bone marrow transplant patients were analysed by both the quantitative PCR (qPCR) and by a previously validated nested nonquantitative PCR (NQPCR). A high level of concordance (90%) was observed between the two assays, although the qPCR assay exhibited slightly greater sensitivity.

Base Sequence↗
PubMed2006

Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide.

The distinction between viable and dead bacterial cells poses a major challenge in microbial diagnostics. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based quantification methods overestimate the number of viable cells in mixed populations or even lead to false-positive results in the absence of viable cells. On the other hand, RNA-based diagnostic methods, which circumvent this problem, are technically demanding and suffer from some drawbacks. A promising and easy-to-use alternative utilizing the DNA-intercalating dye ethidium monoazide bromide (EMA) was published recently. This chemical is known to penetrate only into "dead" cells with compromised cell membrane integrity. Subsequent photoinduced cross-linking was reported to inhibit PCR amplification of DNA from dead cells. We provide evidence here that in addition to inhibition of amplification, most of the DNA from dead cells is actually lost during the DNA extraction procedure, probably together with cell debris which goes into the pellet fraction. Exposure of bacteria to increasing stress and higher proportions of dead cells in defined populations led to increasing loss of genomic DNA. Experiments were performed using Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium as model pathogens and using real-time PCR for their quantification. Results showed that EMA treatment of mixed populations of these two species provides a valuable tool for selective removal of DNA of nonviable cells by using conventional extraction protocols. Furthermore, we provide evidence that prior to denaturing gradient gel electrophoresis, EMA treatment of a mature mixed-population drinking-water biofilm containing a substantial proportion of dead cells can result in community fingerprints dramatically different from those for an untreated biofilm. The interpretation of such fingerprints can have important implications in the field of microbial ecology.

Affinity Labels↗
PubMed2006

Quantitative methylation-sensitive arbitrarily primed PCR method to determine differential genomic DNA methylation in Down Syndrome.

Relative levels of DNA hypermethylation were quantified in DS individuals using a new method based on a combination of methylation-sensitive arbitrarily primed polymerase chain reaction (MS-AP-PCR) and quantification of DNA fragments with the Agilent 2100 bioanalyzer. Four of the DS individuals had low plasma total homocysteine (tHcy) level (4.3 +/- 0.3 micromol/l) and 4 other had high-tHcy level (14.1 +/- 0.9 micromol/l). Eight healthy control individuals were matched to the DS cases for age, sex, and tHcy levels. We have identified and quantified six hypermethylated fragments. Their sizes ranged from 230-bp to 700-bp. In cases and controls, low-tHcy did not affect methylation level of identified fragments, mean methylation values were 68.0 +/- 39.7% and 52.1 +/- 40.3%, respectively. DNA methylation in DS individuals did not change significantly (59.7+/-34.5%) in response to high-tHcy level in contrast to controls (23.4 +/- 17.7%, P = 0.02). Further, the quantitative MS-AP-PCR using this microfludic system is a useful method for determining differential genomic DNA methylation.

Adolescent↗
PubMed1999

Association of p53 genomic instability with the glutathione S-transferase null genotype in gastric cancer in the Portuguese population.

AIMS: p53 gene mutations are the most common genetic changes known to occur in human cancer. In previous studies, the presence of alterations to the p53 gene has been linked to the null phenotype of the glutathione S-transferase mu gene (GSTM1). GSTM1 appears to be part of a protective mechanism against the development of cancers in which environmental chemical carcinogens are involved. To screen for such an association in stomach cancer, p53 allelic loss and genomic instability and GSTM1 genotypes were investigated in gastric tumour DNA samples from 113 patients. METHODS: The polymerase chain (PCR) reaction was used to amplify a (CA) repeat array in the p53 locus; electrophoresis, genotyping, and allele quantification were performed using an automated DNA sequencer and Genescan software. The presence of the GSTM1 gene was determined by means of a differential PCR in which multiple genes were co-amplified in the same reaction tube. RESULTS: Loss of heterozygosity (LOH) of the p53 gene was found in 36 of 87 informative cases and genomic instability was present in eight of 113 cases. Further analysis into histological subtypes and sites of tumours did not show any positive association with p53 loss. An association between the presence of LOH and the GSTM1 null genotype was not seen; however, all the samples with genomic instability of the p53 gene (eight of 113) also showed a GSTM1 null genotype. CONCLUSION: This study does not support the hypothesis of an association between LOH in the p53 gene and the GSTM1 null genotype, but suggests that the GSTM1 null genotype might influence p53 genomic instability.

Alleles↗